Mass spectrometry-based methods for the identification of proteins have become a standard platform in proteomics. The most popular MS-based strategies rely on proteolytic digestion of proteins into peptides before introduction into the mass spectrometer. Digestion of proteins into smaller sized peptides helps to overcome the solubility and handling problems associated with proteins and creates peptide fragments which are easily ionized in the mass spectrometer. Peptide ions are first measured as intact ions, then selected based on their m/z-ratios and subjected to collisionally induced dissociation (CID), in a process known as tandem mass spectrometry (MS/MS). Under the low-energy conditions employed for CID, peptide ions fragment in predictable patterns. Because fragmentation patterns to an extent are predictable, theoretical spectra can be constructed for sequences in protein or nucleotide databases. Computer algorithms can for instance use the CID fragmentation patterns of sample peptides to determine the sequence of the peptide, and this sequence information is used to search against theoretical fragmentation patterns generated from protein and nucleotide databases. Protein identifications are made by finding the best correlation between experimentally derived sequence information and sequences in the database. One of the greatest strengths of tandem mass spectrometry for protein identification is the inherent ability to sequence peptides directly from mixtures. Thus, mass spectrometry allows the direct identification of the individual constituents of protein complexes involved in a wide range of physiological functions. However, if the mixture of peptides is highly complex, it is advantageous to use a separation step prior to analysis to limit the number of peptides the mass spectrometer sees at any given time of the analysis.
One method commonly used to reduce sample complexity prior to introduction into the mass spectrometer is the separation of sample proteins by gel electrophoresis followed by excision of the individual protein spots from the gel and in-gel digestion with a protease (e.g., trypsin). One-dimensional (1D) gels, which separate proteins based on molecular size, provide a low-resolution separation of proteins, but when coupled with tandem mass spectrometry can be used to identify proteins in moderately complex mixtures. For more complex mixtures which are not sufficiently resolved in a 1D separation, a multidimensional separation may be necessary. Multidimensional separations exploit two or more independent physical properties of the proteins or peptides to achieve a higher level of resolution and higher loading capacity than can be achieved in a single dimension. Separation strategies can be selected so that components not separated in the first dimension are separated in the second. Two-dimensional (2D) gels are the most common multidimensional separation technique used to separate proteins in complex mixtures. In this technique, proteins are separated on the basis of their iso-electric point in the first dimension and by their molecular size in the second dimension. The position occupied by a protein on a 2D gel is a reflection of its approximate pI and mass. Although gel-based separations of proteins prior to analysis are effective methods for the analysis of a large number of proteins in complex mixtures, the methods have many shortcomings. One-dimensional gels provide a separation method that is labor intensive, affords limited resolution of proteins, has limited dynamic range, and requires preparation steps that in turn cause decreases in the detection sensitivity by mass spectrometry. To visualize gel-purified proteins so they can be excised and extracted from the gel, the gels must be stained, either by silver staining, Coomassie staining or staining with fluorescent dyes. (Staining gels also allows a quantitative comparison of protein expression, albeit within a limited dynamic range). Two-dimensional gels provide better resolution of proteins but are still labor intensive and have a limited dynamic range. In addition, 2D gels are unable to separate membrane proteins, highly basic proteins or highly acidic proteins under standard conditions.
Gel-based proteomics strategies are rapidly being supplanted by methods that involve peptide separation via high efficiency nano-column liquid chromatography separation techniques directly linked to a mass spectrometer. In these methods, complex protein mixtures are reduced to peptides prior to separation and the total peptide mixture is loaded onto a nanocolumn. The nanocolumn is typically made from fused silica microcapillary tubing that is typically 50-150 μm in inner diameter
and has a tip that has been pulled to an inner diameter of 2-15 μm. For single dimensional separations, the nanocolumn is typically packed with reverse-phase C18 material. Once loaded onto the nanocolumn, peptides are eluted into the ionization source of the mass spectrometer, typically using an HPLC acetonitrile gradient. The gradient is run at very low flow rates, typically 100-400 nL/min and the typical elution time per peptide is 10-30 sec. Stable electrospray at the front of the mass spectrometer's inlet orifice is produced when a voltage of 1.8-2.4 kV is applied to the precolumn liquid-metal interface. As peptide ions enter the mass spectrometer, a survey scan of the intact peptides is obtained. Using data-dependent acquisition, the instrument can be set to automatically monitor the survey scan and select peptides based on pre-set criteria such as intensity, charge state or m/z. The selected peptides are fragmented, and MS/MS spectra are collected. By coupling an LC system with a tandem mass spectrometer and data-dependent scanning, it is possible to distinguish individual proteins in complex mixtures containing several hundred components without additional prior purification or separation steps.
While LC-MS/MS is routinely used to sequence peptides and identify proteins directly from complex mixtures, some samples present complexity beyond the separation capacity of a 1D LC technique. To achieve enhanced separation, gel electrophoresis can be employed to separate intact proteins prior to digestion and loading on the nanocolumn. However, this approach is still encumbered by the shortcomings inherent in gel-based techniques. Recently, a higher-resolution and higher-capacity 2D separation has been achieved with an in-line system using a biphasic nano-column. In this technique, known as multidimensional protein identification technology (MudPIT), a 3-5 cm section of strong cation exchange resin (SCX) is placed directly upstream from the C18 resin in the nanocolumn. The SCX segment has a high loading capacity and is upstream from the RP segment, and thus it acts as a peptide reservoir, storing all peptides until a subset of peptide species is “bumped” to the RP segment with incremental increases in salt in the LC gradient. The dislodged peptides are separated on the RP phase column segment using an acetonitrile gradient and, after re-equilibration, another fraction of peptides is displaced from the SCX stationary phase segment to the RP stationary phase segment with an increase in salt concentration. The iterative process of salt bump followed by RP separation is repeated until the reserve of peptides on the SCX is exhausted. This method greatly increases the number of digested proteins that can be analyzed and enhances the detection of low abundance proteins in the mixture.
MudPIT has been used to identify proteins in samples from a wide variety of sources and has been successfully applied to the identification of posttranslational modifications, as well as the quantitative comparison of protein expression using stable isotope labeling. Two-dimensional separation prior to MS/MS has also been performed in discrete steps by performing an SCX separation sequentially followed by multiple RP separations prior to introduction into the mass spectrometer. In this approach, fractions are collected after the sample is run on an SCX column and each fraction is reduced in volume and loaded onto a reverse phase nano-column for LC-MS/MS analysis. One advantage of discrete separation steps over in-line techniques is that it provides more degrees of freedom for sample manipulation and separation optimization between dimensions. Because each separation phase is independent, there is more flexibility in choices for the composition and sizes of the columns and the length of the gradients. However, the independent application of each phase of separation can result in prohibitively long run times and also lead to severe loss of sample in-between steps.
Two-dimensional LC-MS/MS methods have been shown to be useful for many applications, but complex mixtures of peptides frequently contain salts which can interfere with the interaction of the peptides with the SCX resin. For these samples, on-line desalting can be carried out prior to MudPIT using a solid phase extraction column. Alternatively, desalting can be performed online using a triphasic column which contains a 3 cm segment of C18 packing material directly upstream from the SCX segment. In this technique, peptides are desalted in the first cycle and advanced to the SCX segment where they are subject to multidimensional separation. Optimization of MudPIT is dependent on sample concentration, since the sensitivity of peptide detection in the mass spectrometer is determined by the concentration of the peptide eluted from the column. To optimize detection of the lowest abundance peptides, it is typical to heavily load the column, create small increments in the salt “bumps” to displace peptides from the SCX, and run a long RPLC gradient. In this method, it is common to see highly abundant peptides elute over a number of different salt concentrations. Under most circumstances, this will not interfere with the elution and identification of lower abundant peptides. However, with limited sample quantity, longer gradients should be avoided since they may actually decrease detection sensitivity for low abundance peptides. When sample quantity is limited, it is often useful to optimize conditions using a standard protein mixture of similar concentration. With a well optimized separation it is now possible to identify 1500-2000 proteins from a sample derived from a whole cell lysate.
The success of protein mixture analysis by LC-LC/MS-MS depends on the chromatographic step used to introduce the sample into the mass spectrometer. To achieve good chromatography, high quality nano-bore columns are necessary. Only well-packed nano-bore columns will allow the low flow rates (200-300 nL/min) required for femtomole sensitivity. If the column clogs during sample loading, it is frequently a sign that the sample has not been sufficiently purified prior to loading. Occasionally, salts in the sample can interfere with loading and in these situations the clog can be cleared by briefly immersing the column tip in boiling water. It is possible to use nanocolumns more than once, but extreme care must be taken to make sure all previous sample has been stripped from the column prior to re-loading. Columns must be re-equilibrated after stripping and special care must be taken to make sure the solvent flow is satisfactory.
Setting up the experimental parameters and machine instruction necessary to achieve the desired 2D-LC separation can be quite challenging inasmuch as two separate gradients (one for salt buffers and one for organic phase buffers) have to be interleaved and the action of four pumps must be coordinated with great precision. Small inaccuracies can lead to substantial changes in retention times when experiments are being reproduced. In addition to this, the number of control parameters can be quite substantial, making coding errors likely to occur.
Other disadvantages associated with MudPIT analysis is the uncontrolled flow characteristics of conventional split-flow systems, and the corrosive action of the salty buffers used in the ion exchange chromatographic steps. Salt buffers are highly corrosive and often attack the steel and other materials of which the LC pumps and valves are made. This is obviously an increasing problem with prolonged exposure meaning that the pumps and valves used for generating the salt gradient inevitably will fail or be in need of repair. Also high concentration salt buffers tend to cause problems of particulate matter being generated either by precipitation of the salts themselves or by precipitation of the salts formed from corrosive chemical reactions with the LC pump, valve, and tubing materials.
These and other problems have been solved by the present invention.